Stabilized radiolabeled anti-CD45 immunoglobulin compositions

ABSTRACT

Compositions and methods are described for stabilizing a radio-iodinated monoclonal IgG antibody for up to 17 days against radiolytic decomposition. The stabilized radiolabeled murine antibody binding the CD45 antigen expressed on various forms of lymphomas is useful as a radio-therapeutic and diagnostic agent in the treatment of human malignancies of hematopoietic origin, including lymphomas.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.15/603,817, filed May 24, 2017, which is a continuation-in-part ofInternational Application PCT/US17/21076, filed Mar. 7, 2017, whichclaims priority to U.S. Provisional application No. 62/304,537, filedMar. 7, 2016, the contents of each of which are incorporated herein byreference in their entirety.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to compositions of a radio-iodinatedmurine monoclonal antibody specific for the CD45 antigen, and moreparticularly, compositions comprising the radio-iodinated murinemonoclonal antibody and one or more excipients that stabilize theantibody against radiolytic decomposition.

BACKGROUND OF THE INVENTION

The CD45 antigen is a member of the protein tyrosine phosphatase (PTP)family and is a 180-240 kD trans-membrane glycoprotein. It is also knownas the leukocyte common antigen (LCA), T200, or Ly-5. CD45 plays a keyrole in T-cell and B-cells receptor signal transduction. Differentisoforms of CD45 exist due to variable splicing of its exons. Theseisoforms are very specific to the activation and maturation state of thecell as well as cell type. The various isoforms have the sametrans-membrane and cytoplasmic segments, but different extra-cellulardomains, and are differentially expressed on subpopulations of B- andT-cell lymphocytes. The primary ligands described for CD45 includegalectin-1, CD1, CD2, CD3, CD4, TCR, CD22 and Thy-1.

Depending on which of the alternatively spliced exons (A, B or C) isrecognized, antibodies restricted to recognizing one or the otherisoform have been identified (termed CD45R). In addition, monoclonalantibodies (mAbs) binding an epitope common to all the differentisoforms have also been identified. The mAbs designated CD45RA recognizethe product of exon-A. The mAbs designated CD45RB recognize the productof exon-B. A third type of mAbs termed CD45RO (as exemplified by UCHL1)selectively bind to the 180 kD isoform (without any of the variableexons A, B or C) which is restricted to a subset of cortical thymocytes,activated T cells and memory cells, and is absent on B cells.

In general, all cells of hematopoietic origin, with the exception ofmature erythrocytes and platelets, express CD45. High expression of CD45is seen with most acute lymphoid and myeloid leukemias. Since CD45 isnot found on tissues of non-hematopoietic origin, its specificexpression in leukemia has made it a good target for developingtherapeutics, including radio-immunotherapeutics. For example, CD45 isexpressed at a density of approximately 200,000 to 300,000 sites percell on circulating leukocytes and malignant B cells. One particular¹³¹I-labeled anti-CD45 antibody, BC8, has been explored as a candidateradio-immunotherapeutic alone and in combination with chemotherapy ortotal body irradiation. The use of this ¹³¹I-anti-CD45 antibody for thetreatment of subjects needing bone marrow transplant has also beenexplored.

A number of anti-CD45 antibodies are commercially available. Theseinclude human, mouse, rat, rabbit, canine antibodies and a host ofrelated derived reagents, such as, conjugates with fluorophores,chromophores, biotin and dyes. These also include antibodies derived bythe use of various epitopes, domains and regions of CD45 antigen.Several clones of the species-specific anti-CD45 antibody are alsocommercially available. A list of commercial suppliers of anti-CD45antibodies include the following: eBioscience, Inc., San Diego, Calif.,USA; Novus Biologicals, LLC, Littleton, Colo., USA; Bethyl Laboratories,Inc., Montgomery, Tex., USA; AbD Serotec/Bio-Rad Inc., Raleigh, N.C.,USA; BD Biosciences, San Jose, Calif., USA; AbCam Inc., Cambridge,Mass., USA; Enzo Life Sciences, Inc., Farmingdale, N.Y., USA; R&DSystems Inc., Minneapolis, Minn., USA; EXBIO Praha, A.S., Vestec, CzechRepublic; Life Technologies, Grand Island, N.Y.; and many more. Acomprehensive list of suppliers can be accessed at the followingwebsite:

http://www.biocompare.com/pfu/110447/soids/3537/Antibodies/CD45?vmpi6408=1.

A search at this site for “CD45 antibody” performed on Sep. 25, 2014came up with a total of 3,646 products that are available from 59suppliers. It is evident that a number of anti-CD45 antibodies andrelated reagents are available with their own specific productcharacteristics such as species specificity and antigen heterogeneity,such as epitope or domain specificity.

While large numbers, varieties, and clones of anti-CD45 antibodies areavailable, none is structurally well described. For use as a therapeuticagent, it would be advantageous to properly describe an anti-CD45antibody in order to develop the anti-CD45 antibody into a therapeuticfor the treatment of human indications of hematopoietic malignancies.

Among several clones of the anti-CD45 murine antibody, BC8 recognizesall the human isoforms of the CD45 antigen. The potential applicationsof the BC8 antibody for clinical use, including the treatment of certainkinds of human lymphomas, are well defined in the literature of the past20-25 years. Despite this, the structural composition andcharacterization of this isolated BC8 clone for use as a humantherapeutic has not been adequately described.

Only a partial amino acid sequence from a BC8 clone has been describedas being used in making a single chain construct with streptavidin. Inthis construct, certain regions corresponding to the putative variableregions of the light chain (LC) and heavy chain (HC) were fused togetherwith the streptavidin sequence. Therefore, some 110-120 amino acidstretches related to the LC and HC of BC8 were used in this particularcontext which were indirectly identified by the methods of reversetranscription-PCR (RT-PCR).

As such, the complete structural composition of the BC8 mAb has not beendescribed and characterized.

As indicated above, the BC8 antibody has been shown to bind to all theisoforms of human CD45, and thus provides an excellent target for thedevelopment of therapeutics for certain human malignancies ofhematopoietic origin, including lymphomas. While radiolabelling of BC8for targeted treatment of such malignancies has been explored, none ofthe prior art has been able to achieve radiolabelling efficienciessuitable for its efficient and cost effective use as a therapeutic.Furthermore, BC8 radiolabelled with ¹³¹I has a short half-life (thehalf-life of ¹³¹I is 8.02 days), and must be made and shipped toclinical sites on demand, which impedes its wider use as a therapeutic.

Radio-iodinated antibodies can undergo auto-radiolysis, due to radiationenergy decay and associated free radical mechanisms. This candramatically reduce the antibodies' immune-reactivity, rendering themless effective and thus unsafe for therapeutic use. Moreover, freeradical reactivity towards certain amino acid residues (e.g., Tyr, Trp,His, Met and Cys) in a protein can affect the solution stability of aprotein-based radiopharmaceutical composition. Thus, stabilizingtarget-specific radiopharmaceutical compositions remains a challenge intheir development.

New compositions and methods are needed to stabilize ¹³¹I-anti-CD45immunoglobulins, such as BC8, thereby prolonging shelf life andimproving human therapeutic potential.

SUMMARY OF THE INVENTION

The present invention relates to a stabilized composition comprising anisolated anti-CD45 immunoglobulin (e.g., BC8 mAb clone) in its ¹³¹Iradiolabeled form, and its therapeutic uses. With its ability to bindall the isoforms of the CD45 antigen, BC8 is expected to accumulate atherapeutically high radiation dose specifically and preferentially athigh-density CD45 antigen-bearing cells, such as lymphoma cells.

The present invention further relates to methods of radiolabeling theanti-CD45 antibody in a high radioactivity batch, such as up to 3,000mCi, or even 5,000 mCi, as well as therapeutic and dosimetry doseformulations comprising the radio-iodinated antibody that are stabilizedagainst radiolysis of the antibody.

As such, the present invention provides stabilized radio-iodinatedanti-CD45 immunoglobulin compositions specific for targeting the CD45antigen. Certain formulations may comprise 0.5% to 5.0% (w/v) of anexcipient selected from the group consisting of ascorbic acid,polyvinylpyrrolidone (PVP), human serum albumin (HSA), a water-solublesalt of HSA, and mixtures thereof. Certain formulations may comprise0.5-5% ascorbic acid; 0.5-4% polyvinylpyrrolidone (PVP); and 0.1-2 mg/mLof the radio-iodinated anti-CD45 antibody (e.g., BC8) in 50 mM PBSbuffer, pH 7.

The heavy chain variable domain of the antibody may comprise anN-terminal sequence of SEQ ID NO:12 in combination with at least onecomplementarity determining region (CDR) selected from SEQ ID NO:6, SEQID NO:7, and SEQ ID NO:8; and the light chain variable domain maycomprise an N-terminal sequence of SEQ ID NO:11 in combination with atleast one CDR selected from SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

The heavy chain of the antibody may comprise a sequence of SEQ ID NO:14.

The present invention further provides a composition comprising: ananti-CD45 IgG monoclonal antibody, wherein the anti-CD45 IgG monoclonalantibody is labeled with radio-iodine; and at least one stabilizer(excipient) comprising 0.5 to 5% (w/v) of ascorbic acid, PVP, humanserum albumin (HSA), or water soluble salts of HSA, or combinationsthereof, preferably wherein the stabilizer is added during thepurification of the ¹³¹I-labeled anti-CD45 IgG monoclonal antibodypreparation.

According to certain aspects of the present invention, the stabilizermay be present in a w/v concentration range of 0.5 to 5% in an aqueousformulation comprising phosphate buffered saline, 50 mM pH 7 as aphysiologically acceptable carrier or diluent.

The stabilizer may be ascorbic acid in a w/v concentration of 2.5%included immediately following the quenching of the radio-iodinationreaction and before the purification of the radio-iodinatedanti-CD45-immnuoglobulin. Additionally, 2 to 4% (w/v) HSA may also beincluded along with ascorbic acid at the same step. The purification ofthis mixture using a desalting column may be performed with a pre-cooledmobile phase of PBS buffer (50 mM, pH 7) which is also supplemented withthe 2.5% ascorbic acid or a mixture of 2.5% ascorbic acid plus 2 to 4%HSA (all w/v) as the case may be.

Furthermore, 2% (w/v) PVP may also be included in the purified¹³¹I-anti-CD45-immunoglobulin as yet another stabilizer (excipient)providing stability to the labeled immunoglobulin against radiolysis.

It is also an objective of the present invention to provide, via acocktail of excipients (stabilizers), stability of at least four days(i.e., the first three days at −20° C. and an additional day at roomtemperature) to a therapeutic dose formulation. Preferredstability-enhancing cocktails include w/v 2.5% ascorbic acid and 2% PVP;2.5% ascorbic acid and 4% HSA; and 2.5% ascorbic acid, 4% HSA and 2% PVP(i.e., these cocktails yield immunoglobulin-containing compositionshaving the recited excipient concentrations).

It is yet another objective of the present invention to provide, via acocktail of excipients, stability of at least seven days (i.e., thefirst six days at −20° C. and an additional day at room temperature) toa dosimetry dose formulation. Preferred stability-enhancing cocktailsfor dosimetry dose formulation include w/v 2.5% ascorbic acid and 2%PVP; 2.5% ascorbic acid and 4% HSA; and 2.5% ascorbic acid, 4% HSA, and2% PVP. The stability-indicating assays include an iTLC (instant thinlayer chromatography) assay, size exclusion chromatography-HPLC(SEC-HPLC) and a cell-specific binding-based immunoreactivitymeasurement assay.

It is yet a further objective of the present invention to providemethods for treating a subject (preferably human) afflicted with ahematologic malignancy, comprising administering to the subject aneffective amount of the instant pharmaceutical composition, either aloneor in conjunction with another form of treatment (e.g., a bone marrowtransplant). In one embodiment, the hematologic malignancy is acutemyeloid leukemia, myelodysplastic syndrome, acute lymphoblasticleukemia, Hodgkin's disease or non-Hodgkin's lymphoma. Also envisionedis a method for ablating bone marrow cells in a subject (preferablyhuman) afflicted with leukemia prior to the subject's receiving a bonemarrow transplant, comprising administering to the subject atherapeutically effective amount of the instant pharmaceuticalcomposition. According to certain aspects of the present invention, theleukemia may be acute myeloid leukemia, and the subject may be human,relapsed or refractory, and at least 55 years old.

Also provided by the present invention are aqueous pharmaceuticalcompositions comprising an radio-labeled BC8 antibody; and apharmaceutically acceptable carrier, wherein the radio-labeled BC8antibody comprises a heavy chain having SEQ ID NO:14. The radio-label onthe BC8 may comprise a radiotherapeutic effector molecule, whereinexemplary radiotherapeutic effector molecules include beta emitters suchas, for example, 131I, ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁶Re, or ¹⁸⁸Re, and gamma emitterssuch as, for example, ¹²⁵I or ¹²³I.

Also provided by the present invention are aqueous pharmaceuticalcompositions comprising an ¹³¹I-labeled BC8 antibody; and apharmaceutically acceptable carrier, wherein the ¹³¹I-labeled BC8antibody comprises a heavy chain having SEQ ID NO:14. Methods fortreating a subject afflicted with a hematologic malignancy, includeadministering to the subject an effective amount of this aqueouspharmaceutical composition, either alone or in conjunction with anotherform of treatment.

Still further envisioned is a method for performing dosimetry on asubject (preferably human) afflicted with a hematologic malignancycomprising administering to the subject a dosimetrically effectiveamount of the instant pharmaceutical composition. In this invention,administering is preferably intravenous, and may also be, for example,intramuscular and subcutaneous.

Relating to the present invention are the sequence-relatedcharacteristics of the anti-CD45 immunoglobulin (i.e., BC8) in terms ofthe N-terminal sequences and the CDR sequences of its light chain andthe heavy chain. For the light chain, these sequences are defined as:SEQ ID NO:11 as the N-terminal sequence, SEQ ID NO:3, SEQ ID NO:4 andSEQ ID NO:5 as, respectively, the CDR1, CDR2 and CDR3 regions. For theheavy chain, these sequences are defined as: SEQ ID NO:12 as theN-terminal sequence, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 as,respectively, the CDR1, CDR2 and CDR3 regions. The entire sequence ofthe light and the heavy chains of the anti-CD45 immunoglobulin, aselucidated by the related RT-PCR-derived cDNA construct and LC-MS/MSpeptide mapping approaches, is also provided (SEQ ID NO:13 for the lightchain and SEQ ID NO:14 and 15 for the heavy chain). It is possible thatcertain isomeric amino acid replacements with exact mass, such as Leufor Ile or vice versa, could be allowed in these sequences.

Embodiments disclosed herein define excipient-stabilized radio-iodinatedanti-CD45 immunoglobulin compositions that are useful asradiotherapeutic agents for the treatment of malignancies ofhematopoietic origin.

The objects of the present invention will be realized and attained bymeans of the combinations specifically outlined in the appended claims.The foregoing general description and the following detailed descriptionand examples of this invention are provided to illustrate variousaspects of the present invention, and by no means are to be viewed aslimiting any of the described embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the sequence of the complementarity determining regions(CDRs), framework regions and variable domain sequences of the lightchain (VL) and the heavy chain (VH) of the anti-CD45 mAb. The CDRs arein bold and underlined.

FIG. 2 provides the CDRs and the N-terminal sequence of the light chainand the heavy chain of anti-CD45 mAb.

FIG. 3 provides the entire nucleotide (SEQ ID NO:9) and amino acid (SEQID NO:13) sequence of the light chain of an anti-CD45-immunoglobulin.

FIG. 4A provides the entire nucleotide (SEQ ID NO:10) and actual aminoacid (SEQ ID NO:14) sequence of the heavy chain ofanti-CD45-immunoglobulin.

FIG. 4B provides the entire nucleotide (SEQ ID NO:10) and theoreticalamino acid (SEQ ID NO:15) sequence of the heavy chain ofanti-CD45-immunoglobulin.

FIGS. 5A-5D provide bar graphs of stability test data for a 100 mCi doseof ¹³¹I-anti-CD45 immunoglobulin over a total period of seven days whichincluded two freeze thaw cycles. Size exclusion-High performance liquidchromatography (SEC-HPLC) results showing percent product (FIG. 5A),percent free-¹³¹I (FIG. 5D) and percent fraction of aggregates (FIG.5C), and immunoreactivity results (FIG. 5B) are shown for various timepoints. The time points of this stability study and the formulationexcipient composition were as follows: The radiolabeled anti-CD45antibody initially formulated during the purification process contained2.5% ascorbic acid and 4% HSA (both w/v) and was tested on day 0, andfollowing its storage for 0-3 days at −20° C. On day-3 after thawing, itwas further formulated with 2% (w/v) PVP and once again stored at −20°C. for three additional days. Both stability tests were performed onday-6 followed by storing the formulated product at room temperature foran additional 24 hours after which the stability tests were repeated.

FIGS. 6A-6C provide bar graphs of stability test data of a 1,100 mCitherapeutic dose formulation of ¹³¹I-anti-CD45 immunoglobulin over atotal period of four days which included a freeze thaw cycle. SEC-HPLCresults showing % product (FIG. 6A), % free-¹³¹I and % fraction ofaggregates (FIG. 6C), and immunoreactivity results (FIG. 6B) are shownfor various time points. The time points of this stability study and theformulation excipient composition were as follows. The radiolabeledanti-CD45 antibody initially formulated during the purification processcontained 2.5% (w/v) ascorbic acid, and was formulated with 2% (w/v) PVPand tested on day-0, and following its storage for 0-3 days at −20° C.On day-3, after thawing, the radiolabeled anti-CD45 antibody was storedat room temperature, during which stability was tested at 8 and 24 hours(storage time at room temperature).

FIGS. 7A-7C provide bar graphs of the stability test data of a 13 mCidosimetry dose formulation of ¹³¹I-anti-CD45 immunoglobulin over a totalperiod of seven days which included a freeze thaw cycle. SEC-HPLCresults showing % product (FIG. 7A), % free-¹³¹I and % fraction ofaggregates (FIG. 7C), and immunoreactivity results (FIG. 6B) are shownfor various time points. The time points of this stability study and theformulation excipient composition were as follows. The radiolabeledanti-CD45 antibody initially formulated during the purification processcontained 2.5% (w/v) ascorbic acid and was further formulated with 2%(w/v) PVP and tested on day-0, and following its storage for 0-6 days at−20° C. On day-6 the formulated product was thawed and stored at roomtemperature for an additional 24 hours after which the stability testswere repeated.

FIGS. 8A-8D provide bar graphs of the stability test data of a 150 mCidose of ¹³¹I-anti-CD45 immunoglobulin over a total period of 17 dayswhich included two freeze thaw cycles. SEC-HPLC results showing %product (FIG. 8A), % free-¹³¹I (FIG. 8D) and % fraction of aggregates(FIG. 8B), and immunoreactivity results (FIG. 8C) are shown for varioustime points. The time points of this stability study and the formulationexcipient composition were as follows. The radiolabeled anti-CD45antibody initially formulated during the purification process contained2.5% ascorbic acid and 4% HSA (both w/v) was tested on day 0, andfollowing its storage for 0-14 days at −20° C. On day-14 after thawing,it was then further formulated with 2% (w/v) PVP, tested and once againstored at −20° C. for three additional days. Both the stability testswere performed on day-17 after thawing. It is noteworthy that the 17-dayperiod is almost close to two half-lives of the ¹³¹I isotope (half-life8.02 days).

FIGS. 9A and 9B show a typical radio-elution SEC-HPLC profile of apurified ¹³¹I-anti-CD45-immunoglobulin (FIG. 9A) and the area percent ofeach eluent from peak integration (FIG. 9B).

DEFINITIONS AND ABBREVIATIONS

The following definitions and abbreviations have been used in describingthis invention:

-   -   iTLC—instant thin layer chromatograph    -   IEF—isoelectric focusing    -   SDS-PAGE—sodium dodecyl sulfate-polyacrylamide gel        electrophoresis    -   LC-MS/MS—liquid chromatography-tandem mass spectrometry    -   HSA—human serum albumin    -   PVP—polyvinylpyrrolidone (Povidone)    -   SEC-HPLC—size exclusion chromatography-High performance liquid        chromatography    -   LMWS—low molecular weight species (Free-¹³¹I)    -   HMWS—high molecular weight species (aggregates of antibody        protein)

As used herein, the term “therapeutic dose” refers to an amount ofradiolabeled immunoglobulin sufficient to provide a desired therapeuticradiation dose to the target tissue or organ upon administration to asubject. In one embodiment of the present invention, for example, thisdose ranges between 100 to 1,500 mCi of ¹³¹I radioactivity.

As used herein, the term “dosimetry dose” refers to an amount ofradiolabeled immunoglobulin sufficient to yield an in vivobiodistribution profile for various organs, and a pharmacokineticprofile, in a subject in need of therapy with the immunoglobulin, inorder to help ascertain a subsequent therapeutic dose for the subject.In one embodiment of the present invention, for example, this doseranges between five to 15 mCi of ¹³¹I radioactivity.

As used herein, the terms “antibody” and “immunoglobulin” are usedinterchangeably, and refer, for example, to full-length monoclonalantibodies and polyclonal antibodies. These include, for example, murineantibodies, human antibodies, humanized antibodies, chimeric antibodies,Fab fragments, F(ab′)₂ fragments, antibody fragments with the desiredbiological activity, and epitope-binding fragments of any of the above.Immunoglobulin molecules can be of any type, such as IgA, IgD, IgE, IgGand IgM.

An “epitope” refers to the target molecule site that is capable of beingrecognized by, and bound by, an antibody. For a protein epitope, forexample, this may refer to the amino acids (and particularly their sidechains) that are bound by the antibody. Overlapping epitopes include atleast one to five common amino acid residues. Methods of identifyingepitopes of antibodies are known to those skilled in the art andinclude, for example, those described in Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988).

A “complementarity-determining region”, or “CDR”, refers to amino acidsequences that, together, define the binding affinity and specificity ofthe Fv region of a native immunoglobulin binding site. There are threeCDRs in each of the light and heavy chains of an antibody.

A “framework region”, or “FR”, refers to amino acid sequences interposedbetween CDRs.

A “constant region” refers to the portion of an antibody molecule thatis consistent for a class of antibodies and is defined by the type oflight and heavy chains. For example, a light chain constant region canbe of the kappa or lambda chain type and a heavy chain constant regioncan be of one of the five chain isotypes: alpha, delta, epsilon, gammaor mu. This constant region, in general, can confer effector functionsexhibited by the antibodies. Heavy chains of various subclasses (such asthe IgG subclass of heavy chains) are mainly responsible for differenteffector functions.

As used herein, the term “anti-CD45” describes an antibody comprising atleast one of the heavy chain CDRs and at least one of the light chainCDRs as described in FIGS. 1 and 2.

A “variable domain sequence” is an amino acid sequence that affects thestructure of an immunoglobulin variable domain. This includes all or apart of the amino acid sequence of a naturally occurring variabledomain. For example, the sequence may include other alterations that arecompatible with formation and maintenance of the protein secondary andtertiary structure.

“Fv” is an antibody fragment that contains a completeantigen-recognition and binding site. This region consists of a dimer ofone heavy and one light chain variable domain in tight, non-covalent orcovalent association. Three CDRs of each variable domain interact todefine an antigen-binding site on the surface of the VH-VL dimer.Collectively, the six CDRs confer antigen-binding specificity to theantibody.

“Immunoreactivity” refers to a measure of the ability of animmunoglobulin to recognize and bind to a specific antigen. In oneembodiment, the immunoglobulin binds to its antigen with an affinity(K_(D)) of between 10⁻⁸ M and 10⁻¹⁰ M.

“Purification”, as used herein with respect to an immunoglobulin (orportion thereof), includes, without limitation, the removal of otherimmunoglobulins and related proteins so that the desired immunoglobulin(or portion thereof) is at least 80% pure, at least 90% pure, at least95% pure, at least 98% pure, or at least 99% pure.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing described herein, suitable methods and materialsare described below. In addition, embodiments of the present inventiondescribed with respect to Chothia CDRs may also be implemented usingKabat CDRs.

DETAILED DESCRIPTION OF THE INVENTION

High expression of CD45 antigen is seen on malignant cells associatedwith most acute lymphoid and myeloid leukemias. A density ofapproximately 200,000 to 300,000 sites per cell on circulatingleukocytes and malignant B cells has been reported. Among normal cells,all the cells of hematopoietic origin, with the exception of matureerythrocytes and platelets, express CD45. It is not found on tissues ofnon-hematopoietic origin. Therefore, the leukemia-associated CD45antigen makes it a good target for developing therapeutics, includingradio-immunotherapeutics.

The complete amino acid sequence of the anti-CD45 murine immunoglobulindetermined by the methods described in Example 2 is shown in FIGS. 3 and4A. Estimated CDR regions as determined by homology algorithms are shownin FIGS. 1 and 2. The light chain of the anti-CD45-immunoglobulin wasfound to be a kappa-type chain.

The light chain composed of 218 amino acids contains five cysteineresidues. The heavy chain composed of 445 amino acids contains 12cysteine residues. Both chains fold into immunoglobulin-type domainswith expected cysteine pairs well positioned to establish intra-chaindisulfide bridges. The hinge region on the heavy chain is composed ofthree inter-chain disulfide bonds.

The light chain contains 10 tyrosine residues and the heavy chaincontains 15 tyrosine residues. Therefore, all together, the anti-CD45immunoglobulin contains 50 tyrosine residues which could be targeted asradio-iodination sites.

As such, the anti-CD45 immunoglobulin was labeled with radioiodine usingthe chloramine-T method of radio-iodination as described in Example 3.It was found that an ¹³¹I to immunoglobulin stoichiometry of 20-30mCi/mg consistently afforded about 90% radio-chemical yield followingpurification of the radio-iodinated reaction product. Using thisstoichiometry ratio, the immunoglobulin can be labeled with high levelsof radioactivity (e.g., 300-3,000 mCi, or even 300-5,000 mCi) in asingle batch. As detailed in Examples 3, 10 and 11, thisradio-iodination process utilizing a 30 mCi/mg murine anti-CD45immunoglobulin ratio during radio-labeling resulted in product with >90%radiochemical purity, a radiochemical yield of >90%, and >70%immunoreactivity.

As indicated above, radiolytic damage of ¹³¹I-labelled antibodies hasreduced or even precluded their use as therapeutic agents. It is anobject of the present invention to employ one or more excipients tostabilize ¹³¹I-labelled anti-CD45.

Radical scavengers are employed here as stabilizers to minimizeradiolysis of radiolabeled preparations. These scavengers andstabilizers include, for example, ascorbic acid, gentisic acid, HSA, andpolyvinylpyrrolidone (PVP). Ascorbic acid may be included at from 0.1 to5% (w/v), and may be added post-labeling as a radio-protectant. Gentisicacid may be included at from 0.1 to 0.5% (w/v), or even higher amounts(e.g., 2 to 8%) when included as the sodium salt, and may be addedpost-labeling. HSA may be added at from 2 to 5% (w/v). Individually andin certain combinations, each excipient may slow radiolysis. Inparticular, combinations including ascorbic acid at from 0.1 to 5%(w/v), and HSA at from 2 to 5% (w/v), may be advantageous for slowingradiolytic damage of the ¹³¹I-labelled anti-CD45 of the presentinvention.

PVP is a linear chain water-soluble polymer useful for protectingantibodies against auto-radiolysis. Specifically, the PVP of averagemolecular weight 6-8 kD and a K-17 value (a designator of its viscosity)is the most useful for these purposes. As such, PVP may be included inthe stabilizing formulations disclosed herein at from 0.1% to 6% andeven up to 10% (w/v). PVP may also help to prevent aggregation of theprotein in a composition.

PVP in combination with a secondary stabilization agent, in particular,ascorbic acid or HAS, is most effective in ameliorating auto-radiolysisof radio-peptides in high radioactive fields (>Ci levels). An effectivecombination may include, for example, 0.5-5% (w/v) ascorbic acid and0.5-4% (w/v) PVP, or 0.5-5% (w/v) ascorbic acid, 0.5-5.0% (w/v) HSA, and0.5-4% (w/v) PVP. Ideally, the PVP and ascorbate combination, or PVP,ascorbate and HSA combination, is added immediately following theradiolabeling of the Ab.

In addition to these agents, a variety of other radio-protective agentscan be used, including synthetic thioethers and chromans that providestability through their free radical scavenging and anti-oxidativeproperties.

In additional to chemical means of stabilizing a radiolabeled protein,cryopreservation has also been shown to stabilize the labeledpreparation in enhancing its shelf life. This was exemplified for three¹³¹I-labeled-mAbs where a successful technique of cryopreservation at−70° C. for several days was demonstrated to allow for their shipmentand administration. The immunoreactivity of these cryoprotectedantibodies was fully preserved throughout the course of thisinvestigation (e.g. ˜10 days) with minimal radiation decay, as comparedto samples stored at 4° C. One caveat to this approach, however, is thatan antibody cannot tolerate more than one freeze-thaw cycle.

Thus, another embodiment of the radiolabeling process according to thisinvention is to employ stability-enhancing excipients as early aspossible in the process. For example, excipients such as ascorbic acid(2.5% w/v) may be added alone or in combination with HSA (2% to 4% w/v)to the quenched radio-iodination mixture before the purification of thelabeled product, as detailed in Example 3. Excipients may be added usingpre-cooled concentrated stock solutions.

Purification of the labeled immunoglobulin over a desalting column mayalso be carried out in the presence of excipients, such as the 2.5%ascorbic acid or the mixture of 2.5% ascorbic acid and 2 to 4% HSA (allw/v). For example, the excipients may be added in the pre-cooled elutionbuffer used for purification of the labeled immunoglobulin. The purifiedproduct is therefore collected from the desalting column mixed withthese stability-enhancing excipients. The purification of a 1,000 mCialiquot may be performed on a commercially available sterile SephadexG25 desalting column (GE HiPrep 26/10 desalting column, bed volume 53mL). Similarly, smaller batches such as <200 mCi labeling can bepurified on a commercially available PD10 desalting column (GE PD10column, bed volume 8.6 mL). The use of pre-sterilized columns provides aconvenient method to prepare radiolabeled product doses for humanadministration. While specific column sizes and matrices have beendiscussed, columns of other sizes and bed volumes using similar orequivalent matrices may also be used.

The formulation of the product with stability-enhancing excipients ispreferentially performed using ascorbic acid, PVP and HSA. As such, thepurified radiolabeled product according to this invention may containascorbic acid or a mixture of ascorbic acid and HSA. Further, thepurified product may be formulated with PVP (for example, at 2% w/v) tohelp prevent the formation of aggregates (Example 5).

In addition, a cryopreservation approach to minimizing radiation damageto the high radioactivity labeled immunoglobulin may also be used. Priorcryopreservation schemes have used temperatures as low as −70° C. tostabilize an ¹³¹I-labeled antibody. However, surprisingly, it was foundthat for the radiolabeled immunoglobulin of this invention,cryopreservation at −20° C. gave satisfactory results compatible withtargeted stability and shelf life of four days for therapeutic doseformulations and eight days for the dosimetry dose formulations.Furthermore, as described in Examples 8-10, the use of thiscryopreservation surprisingly allowed two freeze-thaw cycles whilemaintaining the integrity and stability of the formulated ¹³¹I-labeledanti-CD45 immunoglobulin of this invention.

Thus, the compositions, stabilizing excipient mixtures andcryopreservation methods of the present invention have been found toprovide excellent stabilization of the radiolabelled antibody, providingintact antibodies having immunoreactivity of greater than 70%, greaterthan 80%, and even greater than 90%.

Various stability-indicating tests performed (Example 7) with thetherapeutic and dosimetry dose formulations suggest that, for example,the combination of 2.5% ascorbic acid and 2% PVP (both w/v) asexcipients, along with cryopreservation at −20° C., provides goodstability of four days and eight days, respectively, for the therapeuticdose and the dosimetry dose formulations. In both instances, theterminal day of storage is at ambient temperature.

Each of the foregoing embodiments that provides excipient-stabilizedradio-iodinated anti-CD45 immunoglobulin compositions may find use as aradiotherapeutic agent for treating malignancies of hematopoieticorigin.

The following examples of this invention are provided to illustrate itsvarious aspects and by no means are to be viewed as limiting any of thedescribed embodiments. A different combination of these embodiments maybe used to achieve similar characteristics of the formulatedradio-iodinated immunoglobulins of this invention. For example, theamounts of the excipients used may be varied and the temperature ofstorage may be changed to 2-8° C. or even room temperature. This may beadaptable to formulations intended for immediate use or use within ashort period of 1-2 days.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Inaddition, the materials, methods and examples are illustrative only andnot intended to limit the scope of the present invention.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 shows the amino acid sequence of the variable domain of thelight chain of anti-CD45 murine immunoglobulin BC8.

SEQ ID NO:2 shows the amino acid sequence of the variable domain of theheavy chain of anti-CD45 murine immunoglobulin BC8.

SEQ ID NO:3 shows the amino acid sequence of CDR1 of the light chain ofanti-CD45 murine immunoglobulin BC8.

SEQ ID NO:4 shows the amino acid sequence of CDR2 of the light chain ofanti-CD45 murine immunoglobulin BC8.

SEQ ID NO:5 shows the amino acid sequence of CDR3 of the light chain ofanti-CD45 murine immunoglobulin BC8.

SEQ ID NO:6 shows the amino acid sequence of CDR1 of the heavy chain ofanti-CD45 murine immunoglobulin BC8.

SEQ ID NO:7 shows the amino acid sequence of CDR2 of the heavy chain ofanti-CD45 murine immunoglobulin BC8.

SEQ ID NO:8 shows the amino acid sequence of CDR3 of the heavy chain ofanti-CD45 murine immunoglobulin BC8.

SEQ ID NO:9 shows the nucleotide sequence of the light chain ofanti-CD45 murine immunoglobulin BC8.

SEQ ID NO:10 shows the nucleotide sequence of the heavy chain ofanti-CD45 murine immunoglobulin BC8.

SEQ ID NO:11 shows the amino acid sequence of N-terminus of the lightchain of anti-CD45 murine immunoglobulin BC8.

SEQ ID NO:12 shows the amino acid sequence of N-terminus of the heavychain of anti-CD45 murine immunoglobulin BC8.

SEQ ID NO:13 shows the predicted amino acid sequence of the light chainof anti-CD45 murine immunoglobulin BC8.

SEQ ID NO:14 shows the predicted amino acid sequence of the heavy chainof anti-CD45 murine immunoglobulin BC8.

SEQ ID NO:15 shows the actual amino acid sequence of the heavy chain ofanti-CD45 murine immunoglobulin BC8 as determined by amino-acidsequencing.

EXAMPLES Example 1 Production of Anti-CD45 Immunoglobulin BC8

The murine anti-CD45 mAb BC8 was prepared from a hybridoma (ATCC No.HB-10507) that was initially developed by fusing mouse myeloma NS1 cellswith spleen cells from a BALB/C mouse hyperimmunized with humanphytohemagglutinin (PHA)-stimulated mononuclear cells. The originalfused cells, after screening for microbial contaminations, were culturedusing the JRH-Biosciences EXCell 300 medium supplemented with 1-2% FetalBovine Serum (FBS).

The hybridoma cell line was adapted for culture in a serum-free culturemedium. Briefly, the cells in culture were slowly and gradually weanedof the serum albumin using the combo medium supplemented with glutamine,cholesterol, insulin and transferrin. The cells were then grown in up to500 L scale to a density of >1 e6 cells/mL The medium was harvested andprocessed for the purification of the anti-CD45 antibody using acombination of cation exchange chromatography, Protein-A chromatography,and anion exchange membrane separation. The purified antibody wasconcentrated by nano-filtration (30 kD cutoff). The concentration of thepurified product was measured at 5.2 mg/ml and was stored at 2-8° C.

The purified antibody was characterized by SDS-PAGE, IEF and SEC-HPLCtechniques. A single product peak (99.4%) was recorded with SEC-HPLCwith about 0.6% aggregates. The non-reducing SDS-PAGE showed the band at186.55 kD for the antibody. The SDS-PAGE under reduced conditionsconfirmed the presence of the light and the heavy chains (99.9%together).

Example 2 Sequencing of the Anti-CD45-Immunoglobulin BC8

DNA Sequence: Total RNA was isolated from the hybridoma cells followingthe technical manual of Trizol® Reagent. The total RNA was analyzed byagarose gel electrophoresis and was reverse transcribed into cDNA usingisotype-specific anti-sense primers or universal primers following thetechnical manual of PrimeScript™ 1st Strand cDNA Synthesis Kit. Theantibody fragments of VH, VL, CH and CL were amplified and wereseparately cloned into a standard cloning vector using standardmolecular cloning procedures. Colony PCR screening was performed toidentify clones with inserts of correct sizes. More than five singlecolonies with inserts of correct sizes were sequenced for each antibodyfragment. The complete nucleotide sequence of the light and the heavychains are shown in FIGS. 3 and 4A.

Protein Sequencing by LC-MS/MS: The anti-CD45-immunoglobulin wassequenced using the mass spectrometry peptide mapping approach. Theanti-CD45-immunoglobulin was de-glycosylated, reduced and digested withindividual enzymes; trypsin, Lys-C and chymotrypsin. The peptidefragments were then analyzed by the LC-coupled mass spectrometrytechnique using the MS/MS fragmentation analysis approach. In theLC-MS/MS peptide mapping-based sequence, certain isomeric amino acids ofthe same mass may be mistaken for one another. For example,interpretation between a Leu and Ile can be difficult. Thenucleotide-based sequence was used to correct for such ambiguities.

The complete predicted protein sequences of the light and the heavychain, based on the DNA sequence and standard codon usage, are shown inFIGS. 3 and 4B. Protein sequencing of the heavy and light chains of theBC8 antibody showed that the actual amino acid sequence differs fromthat predicted by the DNA sequence by only a single amino acid in theheavy chain. The complete actual sequence of the heavy chain, based onthis protein sequencing, is shown in FIG. 4A. The codon which codes forthe amino acid at position 141 predicts an ASN-141 and not the actualASP-141 found by protein sequencing.

This type of post-translational modification, deamination, may depend onthe cellular environment and, in some cases, has been postulated to berelated to protein age (e.g., may provide a signal for proteindegradation). The fact that other deaminated amino acids were notidentified, however, may be indicative of an important and specific rolefor ASP-141. At the very least, ASP-141 may be in an exposed oraccessible region on the folded protein. That is, ASN-141 may be solventaccessible and reside within a conformationally flexible region of theantibody. The effect of deamination on the biological activity of theBC8 antibody may be determined from the results of human clinicaltrials.

Example 3 Radio-Iodination of Anti-CD45 Immunoglobulin BC8 and itsPurification in the Presence of Ascorbic Acid

One mg of anti-CD45 immunoglobulin was labeled with 20 to 30 mCi of¹³¹I—Na (30 mCi) in the presence of chloramine-T (23 micrograms) in PBSbuffer (pH 7.2). The reaction was quenched with the addition of aqueoussodium thiosulfate (69 micrograms) and diluted with cold NaI (1 mg).Immediately following, a concentrated ascorbic acid solution made in 50mM PBS (pH 7) was added to achieve 2.5% (w/v) ascorbic acid strength inthe quenched reaction mixture. Labeling reactions up to 3,000 mCi perbatch were successfully performed using this method.

The labeled product was purified by gel filtration on a sterile,pre-packed commercially available Sephadex G25 column (GE HiPrep 26/10column, bed volume 53 mL) using PBS (50 mM, pH 7) mobile phasesupplemented with 2.5% (w/v) ascorbic acid to stabilize the radiolabeledproduct. Up to 1,000 mCi reaction volume was purified on a singlecolumn. The product was collected in the five to 35 mL elution volumefrom the column.

The radio-iodinated reaction batches of <200 mCi could be purified in asimilar fashion on a smaller desalting column (GE PD10 column, bedvolume 8.6 mL).

Example 4 Radio-Iodination of Anti-CD45 Immunoglobulin BC8 and itsPurification in the Presence of Ascorbic Acid and HSA

The labeling and purification were performed essentially as described inExample 3, except that 2% or 4% (w/v) HSA was also added along with 2.5%(w/v) ascorbic acid to the quenched reaction as well as in the elutionbuffer during the purification process.

Example 5 Formulation of ¹³¹I-Anti-CD45 Immunoglobulin BC8 to Stabilizeit Against Auto-Radiolysis

Aliquots of the purified product were formulated at once for stabilitytest studies under various storage conditions and durations. The storagetest conditions included −20° C., 2-8° C., room temperature and acombination thereof. The duration of storage was three days to 15 days,followed by one additional terminal day at room temperature. The −20° C.conditions were intended to stabilize the formulated product againstauto-radiolysis. The terminal 24 hr room temperature condition was totest for stability of the product during its administration to humansubjects.

The purified ¹³¹I-anti-CD45-immunoglobulin product contained 2.5% (w/w)ascorbic acid (as in Example 3) and additionally 2 or 4% (w/v) HSA (asin Example 4). In either case, the product was further formulated bysupplementing it with 2 or 4% (w/v) PVP. The PVP was added for itsinfluence in preventing the formation of protein aggregates. In thisinvention, ¹²⁵I and ¹²³I are also envisioned for use in radiolabelingAnti-CD45 Immunoglobulin (e.g., BC8).

All of the formulated products were assayed for stability andimmunoreactivity following the above mentioned stability test paradigms.

Example 6 Stabilized Formulations of ¹³¹I-Anti-CD45 Immunoglobulin BC8For Dosimetry and Therapeutic Applications

The purified product was formulated at once into unit doses fordosimetry and therapeutic applications. Appropriate aliquots (mCi) ofthe purified ¹³¹I-anti-CD45 immunoglobulin were formulated into singledoses, each having a 45 mL dose volume. The radioactivity drawn in thealiquots was calculated for the desired calibration time (CT) of threedays for the therapeutic dose and seven days for the dosimetry dose. Asingle lot of purified ¹³¹1-BC8 was used to make a combination of singleunit dosimetry and therapeutic doses.

To make a dosimetry dose formulation, a 6 to 12 mCi aliquot (for CT ofseven days) was formulated to a 50 mL dose volume, by combining it withappropriate amounts of “cold” (i.e., unlabeled) anti-CD45 immunoglobulin(35 mg final amount of the combined labeled and unlabeled antibodyfractions in 45 mL dose volume for a 70 kg subject), 2.5% (w/v) ascorbicacid and 2% (w/v) PVP. A 45 mL portion was filled in the dose vial. Theremaining 5 mL was filled in a vial designated for QC tests. Afterformulation, the doses can be stored at −20° C. for up to seven days foruse within this time period. For human administration, a dose receivedat −20° C. could be allowed to stand at room temperature for one to twohours to thaw it. It may then be infused into the patient within thenext 22 hours.

The therapeutic dose formulations were made patient-specific both interms of dose-associated radioactivity and the amount of the anti-CD45immunoglobulin. The radioactivity dose amount as prescribed by theclinical investigator was formulated by matching it with the calculatedamount of total anti-CD45 immunoglobulin (cold anti-CD45 immunoglobulinplus ¹³¹I-radiolabeled anti-CD45 immunoglobulin fraction) adjusted forthe weight of the patient to deliver 0.5 mg/kg BC8 antibody dose in a 45mL dose volume. A therapeutic dose formulation was also adjusted to afinal 2.5% (w/v) ascorbic acid and 2% (w/v) PVP. Using these parameters,a 50 mL volume of the therapeutic dose was made, of which 45 mL aretransferred to a dose vial. The remaining 5 mL of the formulation isplaced in a vial designated for QC tests. The formulated dose could bestored at −20° C. for up to three days for administering within thisperiod. A dose received by the clinical site at −20° C. could be allowedto stand at room temperature for one to two hours to thaw it. It maythen be infused into the patient within next 22 hours.

Example 7 Analytical Methods For Testing the Stability of¹³¹I-anti-CD45-Immunoglobulin BC8

The radiochemical purity and immune-reactivity of the ¹³¹I-labeled BC8are two important measurable analytical parameters that determine itssuitability for clinical use as a dosimetry agent and a therapeuticagent. Various analytical methods employed to establish theserequirements are described herein.

(A) Radiochemical Incorporation by Instant Thin-Layer Chromatography(iTLC).

Silica-impregnated iTLC strips of dimension 10.5×1.0 cm (Gelman SciencesInc., Ann Arbor, Mich.; or Varian ITLC-SG; SG10001) were employed forthis test. Each strip was marked with pencil lines at 1.5, 6.0 and 6.5cm from one edge. The strip was spotted on the 1.5-cm line with about25-50 μCi of the ¹³¹I-BC8 preparation (generally, 1-5 μL). The strip wasthen set in a 15-mL plastic centrifuge tube containing about 2 mL ofnormal saline (or PBS buffer, or sodium acetate buffer) to serve as themobile phase. The iTLC strip was developed until the mobile saline phasehad reached the top of the strip. The strip was taken out of the tubeand cut at the 6.0- and 6.5-cm lines. The activities of the top, middleand bottom sections were measured in a dose calibrator. Theradiochemical incorporation of ¹³¹I to the immunoglobulin protein wascalculated as the activity of the bottom section divided by the totalactivity in all three sections. The acceptance criterion was RCP≥90%.Significant (≥5%) activity in the middle section would be indicative ofstreaking and require a re-test.

${{Radiochemical}\mspace{14mu}{incorporation}\mspace{14mu}(\%)} = \frac{( {{Bottom}{\mspace{11mu}\;}{counts}} )}{( {{Bottom} + {Middle} + {{Top}\mspace{14mu}{count}}} )}$

For the purified ¹³¹I-anti-CD45-immunoglobulin preparations, this methodshowed 98-100% of the radioiodine in the immunoglobulin with an almostimmeasurable amount of radioactivity in the top half of the iTLC strip.The free ¹³¹I, if present, is carried to the top of the strip by themobile phase. This test, therefore, routinely established that duringthe purification of the ¹³¹I-anti-CD45 immunoglobulin by desaltingchromatography immediately following the radio-iodination reaction, thefree iodine was effectively removed from the labeled product.

(B) Radiochemical Purity Test (RCP) by Size ExclusiveChromatography-High Pressure Liquid Chromatography (SEC-HPLC).

The HPLC-SEC assay was performed using a YMC-pack-Diol 200 column, usinga 50 mM phosphate, 150 mM NaCl mobile phase at pH 7.2 and a flow rate of0.5 mL/min. The column was first rinsed with 10 mM phosphate, pH 7.0 forat least 60 minutes. It was then equilibrated with the mobile phase foranother 60 minutes before injection of the sample. Samples were run for45 minutes, with blanks (water) injected between each sample to ensurethere was no overlap of peaks from one sample to the next. A referencestandard was also tested to compare the main peak elution time.

The SEC using this column allows for the separation of the ¹³¹I-antiCD45 immunoglobulin protein into the product (monomeric) and aggregatedfractions, in addition to the free unconjugated radio-iodine and othersmaller protein degradation fragments. The radiochemical purity of¹³¹I-anti-CD45 immunoglobulin using HPLC-SEC is obtained using anin-line flow gamma radioactivity detector. A typical profile andintegration results are shown in FIGS. 9A and 9B.

The data for a typical run were obtained as the % radioactivityassociated with the ¹³¹I-anti CD45-immunoglobulin product (monomer), thehigh molecular weight species or aggregated protein, and the free-¹³¹I.For the purified ¹³¹1-anti CD45-immunoglobulin preparations, this methodconsistently showed (a) >95% monomer peak corresponding to the desiredlabeled product; (b) <5% aggregates; and (c) <5% of low molecular-weightimpurities, including the free ¹³¹I. A comparison of these threecritical quality attributes among various formulations and stabilityconditions was made to understand the effectiveness of a formulation andits stability over a period of time and temperature conditions.

(C) Immunoreactivity Assays.

In these assays, Raji, Ramos, or CytoTrol (Beckman Coulter) cellsexpressing cell surface CD45 antigen were used to determine specificbinding of ¹³¹I-anti-CD45-immunoglobulin to CD45 antigen-positive cells.The CytoTrol cells were lyophilized human lymphocytes isolated fromperipheral blood that exhibit CD45 surface antigen and were preferreddue to their commercial availability (Beckman Coulter) and consistency.

(i) Direct binding assay: An aliquot of the ¹³¹I-anti-CD45immunoglobulin was diluted to 30 ng/mL. A 2-mL micro-centrifuge vial wascharged with 100 μL of cells (2.5×10⁷CytoTrol cells) and 150 μL of thediluted ¹³¹I-anti-CD45 immunoglobulin (4.5 ng protein). For themeasurement of total activity termed “Applied Total”, another vial wascharged with only the ¹³¹I-BC8 solution (150 μL). The assay wasperformed in triplicate. The cell-protein mixtures were incubated on arocker at 37° C. for one hour, and then centrifuged at 2,000 rpm for twominutes. A specific volume of the supernatant from each vial (188 μL)was transferred by pipette to empty micro-centrifuge vials. The activityin each vial of supernatant (as well as in the “Applied Total” vials)was measured with a NaI well counter (Capintec), counting each vial forone minute. The % binding of ¹³¹I-BC8 to CytoTrol cells was thencalculated as:

${{Binding}\mspace{14mu}(\%)} = \frac{ {( {{Applied}\mspace{14mu}{Total}\mspace{14mu}{activity}} ) = {( \frac{4}{3} )( {{supernatamt}\mspace{14mu}{activity}} \rbrack}} )}{( {{Applied}\mspace{14mu}{Total}\mspace{14mu}{activity}} )}$

A >70% specific binding was considered an acceptable level ofimmunoreactivity for the ¹³¹I-radiolabeled product.

(ii) ELISA competition assay: A cell basis ELISA competition assay wasalso used to measure ¹³¹I-anti-CD45-immunoglobulin BC8 binding affinityto CytoTrol cells. In this assay, various concentrations of¹³¹I-anti-CD45-immunoglobulin were made to compete with the binding of afixed concentration of CD45-FITC antibody to the CytoTrol cells. TheCD45-FITC antibody is a fluorescein isothiocyanate (FITC)-labeledmonoclonal antibody that reacts with all isoforms of human CD45. Theincreasing concentrations of ¹³¹I-anti-CD45-immunoglobulin displaced theCD45-FITC bound to CytoTrol cells. The binding activity was thenmeasured in terms of fluorescence of CD45-FITC bound on CytoTrol cells.The immunoreactivity of ¹³¹I-anti-CD45-immunoglobulin was defined as theconcentration of ¹³¹I-anti-CD45-immunoglobulin at which ≥80% cellbinding of CD45-FITC antibody to CytoTrol cells was displaced.

(iii) Flow Cytometry Assay: A Flow Cytometry assay can also be used toevaluate the immunoreactivity of the ¹³¹I-anti-CD45-immunoglobulinformulations. The specific binding to the CD45 cell surface antigen (onCytoTrol or Raji Cells) was measured in terms of an arithmetic mean,geometric mean, or median fluorescence intensity (MFI). The MFI shoulddemonstrate radiolabeled anti-CD45-immunoglobulin binding: ≥50% of thepositive control antibody with known binding ability. The positivecontrol antibody in this assay could be the native (unlabeled) antiCD45-immunoglobulin.

Example 8 Stability Studies on a 100 mCi ¹³¹I-Anti-CD45-ImmunoglobulinFormulated With (w/v) 2.5% Ascorbic Acid, 4% HSA and 2% PVP

A 100 mCi aliquot of the initial labeled product containing 2.5%ascorbic acid and 4% HSA (prepared by the methods of Examples 4 and 5)was stored frozen at −20° C. for three days, thawed and assayedaccording to methods of Example 7. It was then formulated to include 2%PVP and stored at −20° C. for three days, then thawed, assayed and leftat ambient temperature for one day. Day 7 assays were also performed forcomparisons.

The assay results shown in FIGS. 5A-5D describe a very high stability ofthe labeled and formulated product and resistance to degradation by twofreeze/thaw cycles during the entire stability testing process. Themonomeric protein bound activity at all the test points over the entireseven-day period, which include the terminal day 7 at room temperature,were well over 90% with acceptable immunoreactivity of >80%. Theaggregates (HMWS) and the Free-I (LMWS) were also well contained; <3%and <4.5% respectively. Considering the half-life of ¹³¹I (8.02 days),these 7-day stability results are remarkable for the exemplaryexcipient-stabilized formulation.

Example 9 Stability Testing of Three Therapeutic Dose Formulations Using(w/v) 2.5% Ascorbic Acid and 2% PVP For Over a 4-day Period

TABLE 1 Summary of key stability parameters of therapeutic doseformulations studied Formulation iTLC IR (% Cell Binding) SEC-HPLCexcipient T = 0-3 T = 0-3 T = 0-3 Labeling Therapeutic AA PVP HSA day atT = day day at T = day day at T = day Batch Formulation % % % T = 0 −20°C. 4 at RT T = 0 −20° C. 4 at RT −20° C. 4 at RT 1,000   895 mCi 2.5 2 —99.64 98.93 97.99 83.3 79.1 78.8 Prod. 96.47 94.27 92.83 mCi formulationFree 1.25 3.38 5.05 Run-3 Aggr. 2.28 2.35 2.13 3,000 1,182 mCi- 2.5 2 —99.60 98.90 98.80 80.9 80.8 75.9 Prod. 96.69 92.83 92.47 mCi Th-A Free1.14 3.89 5.59 Run-4 formulation Aggr. 2.18 3.28 1.94 3,000 1,133 mCi2.5 2 2% 99.59 98.30 98.30 83.2 79.9 76.2 Prod. 96.86 93.94 91.21 mCiTh-B: Free 1.07 3.72 6.21 Run-4 formulation Aggr. 2.06 2.34 2.57AA—acetic acid; PVP—polyvinylpyrrolidone; HSA—human serum albumin

Aliquots (895, 1,133 and 1,182 mCi) from 1,000 to 3,000 scale labelingruns performed according to the methods of Example 3, or Examples 3 and4 combined, were formulated with 2% (w/v) PVP. Table 1 details theseindividual therapeutic dose formulations and stability testing resultsusing a protocol of 3-day storage at −20° C. followed by an additionalday at room temperature. The stability-indicating assays were performedaccording the methods of Example 7. All the test results at the end ofday 4 showed >90% monomeric product fraction with a maximum of 6.21% ofFree-I and 3.3% aggregates, signifying high stability of the product.The immunoreactivity of >75% at the day 4 time point was also excellent.FIGS. 6A-6C show histograms of the 1,133 mCi therapeutic formation ofthis set.

Example 10 Stability Testing of Seven Dosimetry Dose Formulations Using(w/v) 2.5% Ascorbic Acid and 2% PVP For up to an 8-day Period

TABLE 2 Summary of key stability parameters of dosimetry doseformulations studied Formulation iTLC IR (% Cell Binding) SEC-HPLC Scaleof excipient T = 0-7 T = 0-7 T = 0-7 Labeling Dosimetry AA PVP HSA dayat T = day day at T = day day at T = day Batch Formulation % % % T = 0−20° C. 8 at RT T = 0 −20° C. 8 at RT T = 0 −20° C. 8 at RT   300 mCiA-12 mCi 2.5 2 4 99.60 99.98 98.39 85.3 80.1 77.9 Prod. 95.50 95.99 96.2formulation Free 2.70 1.85 2.11 Aggr. 2.70 2.16 1.69   300 mCi B-18 mCi2.5 2 4 99.60 99.78 98.53 85.3 80.4 77.2 Prod. 97.50 95.65 95.61formulation Free 1.06 2.07 2.28 Aggr. 1.44 2.28 2.11   300 mCi 12 mCi2.5 2 4 96.85 97.26 96.47 77.9 79.9 76.1 Prod. 96.85 97.26 96.47formulation Free 1.32 1.29 1.99 Aggr. 1.82 1.45 1.54 1,000 mCi A-11.56mCi 2.5 2 — 99.75 98.91 98.83 83.8 80.9 77.3 Prod. 96.88 96.39 (d3)96.54 (d4) formulation (d = 3, (d = 4, (d3) (d4) Free 0.92  1.25 (d3) 1.81 (d4) −20° C.) RT) Aggr. 2.20  2.36 (d3)  1.65 (d4) 1,000 mCiB-11.25 mCi 2.5 2 — 99.75 96.08 na 83.8 78.7 na Prod. 96.88 96.11 Naformulation Free 0.92 1.43 Na Aggr. 2.20 2.47 Na 3,000 mCi Ds-A-15.032.5 2 — 98.97 98.80 98.3 80.4 81.2 80.7 Prod. 96.68 95.38 (d3)  96.6(d4) mCi (d3) (d4) (d3) (d4) Free 1.25  1.58 (d3)  1.33 (d4) formulationAggr. 2.08  2.41 (d3)  2.07 (d4) 3,000 mCi Ds-B-13.78 2.5 2 — 98.9797.72 97.12 80.4 78.4 71.5 Prod. 96.68 96.88 (d6) 96.76 (d7) mCi (d6)(d7) Free 1.25  1.85 (d6)  1.41 (d7) formulation Aggr. 2.08  1.26 (d6) 1.82 (d7) RT is room temperature. The d3, d4, d6 and d7 stands for day3 to day 7

Seven 11 to 18 mCi aliquots from 300 to 3,000 scale labeling runsperformed according to the methods of Example 3 or Example 3 and 4combined were formulated with 2% (w/v) PVP. Table 2 describes theseindividual dosimetry dose formulations and stability testing resultsusing a protocol of 3- or 7-day storage at −20° C. followed by anadditional day at room temperature. The stability-indicating assays wereperformed according the methods of Example 7. All test results at theend of day 4 or day 8 showed >95% monomeric product fraction with amaximum of 2.8% of Free-I and 2.7% aggregates, signifying high stabilityof the formulated product. The immunoreactivity of >75% at the day 4time point and >70% at day 8 time point was also well within acceptablerange. FIGS. 7A-7C show histograms of the 13.78 mCi dosimetry doseformation of this set as a representative example.

Example 11 Stability Studies on a 150 mCi ¹³¹I-anti-CD45-ImmunoglobulinFormulated With (w/v) 2.5% Ascorbic Acid and 4% HSA as Well as 2.5%Ascorbic Acid, 4% HSA and 2% PVP

A 150 mCi aliquot of the initial labeled product containing 2.5%ascorbic acid and 4% HSA (prepared using the methods of Examples 3 and4) was stored frozen at −20° C. for 14 days. It was thawed, assayedusing methods of Example 7, and further formulated with the inclusion of2% PVP. This fully formulated product was assayed and again deposited at−20° C. for three additional days. It was finally thawed on day 17 andassayed. This entire stability study included two freeze-thaw cycles,and the combined 17-day stability period is more than two half lives ofthe ¹³¹I isotope.

The results shown in FIGS. 8A-8D underscore the stability of the fullyformulated sample for over 17 days (more than double the ˜eight-dayhalf-life of ¹³¹I). The monomeric protein-bound activity at all the testpoints over the entire 17-day period was >90%, with an immunoreactivityof >70%. The aggregate fraction (HMWS) and the Free-I (LMWS) were alsowell contained at <1% and <6%, respectively. The immunoreactivity of thesample frozen for 14 days was recorded to be >85%. These assay resultsfor long-term storage of the labeled product (total 17 days) indicategood overall stability of the labeled product and its resilience toprolonged freezing for two weeks followed by two thawing cycles.

Example 12 Additional Embodiments of ¹³¹I-BC8 Formulations

In one embodiment, each ¹³¹I-BC8 dosimetric dose contains a volume of45±1 mL with 31.5-38.5 mg of BC8, composed of 131I-labeled BC8 andunlabeled BC8, in a solution of 50 mM sodium phosphate buffered saline(0.9%) (pH 7.0) containing 25 mg/mL ascorbic acid, and 20 mg/mL PVP. Thedosimetric dose contains 10 (or 10-15) mCi of ¹³¹I-BC8, and is intendedfor complete infusion during i.v. administration.

In another embodiment, each ¹³¹I-BC8 therapeutic dose contains a volumeof 45±1 mL with 6.66-45.0 mg of BC8, composed of ¹³¹I-labeled BC8 andunlabeled BC8, in a solution of 50 mM sodium phosphate buffered saline(0.9%) (pH 7.0) containing 25 mg/mL ascorbic acid, and 20 mg/mL PVP. Thetherapeutic dose contains 200-1,350 mCi of ¹³¹I-BC8, depending onpatient-specific needs, and is intended for complete infusion duringi.v. administration. The therapeutic dose does not exceed a ratio of30:1 of mCi to mg of antibody.

The therapeutic dose activity level is calculated based on gamma cameraimages following the dosimetric administration using Medical InternalRadiation Dose (MIRD) method and Organ Level Internal Dose Assessment(OLINDA) software to calculate absorbed dose per unit of administeredactivity to project levels not exceeding 24Gy of activity to any normalorgan and 48Gy of activity to the bone marrow.

In one embodiment of these ¹³¹I-BC8 formulations (i.e., doses), theformulations are prepared and stored in the form of batches, wherebyeach batch is ultimately divided into individual doses. So, for example,one batch might contain between 1-2 Ci, 1-3 Ci, 2-3 Ci, 1-4 Ci, 2-4 Ci,1-5 Ci, 2-5 Ci, 3-5 Ci, 4-5 Ci, 1-10 Ci, 2-10 Ci, 3-10 Ci or 5-10 Ci. Ina 3 Ci batch, for example, the molar ratio of ¹³¹I to antibody is 0.28,the molar ratio of ascorbate to ¹³¹I is 53.83, and the molar ratio ofascorbate to antibody is 415.64. In a 2 Ci batch, for example, the molarratio of ¹³¹I to antibody is 0.18, the molar ratio of ascorbate to ¹³¹Iis 80.74, and the molar ratio of ascorbate to antibody is 277.09. By wayof further example, in a 3 Ci batch, the molar ratio of ascorbate to¹³¹I is below 60, below 55, below 50, below 45, below 40, below 35,below 30, below 25 or below 20; and/or the molar ratio of ascorbate toantibody is below 500, below 450, below 400, below 350, below 300, below250 or below 200.

REFERENCES

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What is claimed is:
 1. A method for preparation of a stabilizedradiotherapeutic composition for administration to a subject, the methodcomprising: radiolabeling an anti-CD45 IgG monoclonal antibodycomprising a light chain comprising light chain complementaritydetermining regions 1 through 3 respectively having the amino acidsequences set forth in SEQ ID NO:3, SEQ ID: NO:4 and SEQ ID NO:5 and aheavy chain comprising heavy chain complementarity determining regions 1through 3 respectively having the amino acid sequences set forth in SEQID NO:6, SEQ ID NO:7 and SEQ ID NO: 8 with ¹³¹I to form an ¹³¹I-labeledanti-CD45 IgG monoclonal antibody, purifying the ¹³¹I-labeled anti-CD45IgG monoclonal antibody in the presence of ascorbic acid, andsupplementing the purified ¹³¹I-labeled anti-CD45 IgG monoclonalantibody with polyvinylpyrrolidone (PVP), wherein the stabilizedradiotherapeutic composition comprises 0.5-5.0% (w/v) ascorbic acid and0.5-5.0% (w/v) PVP.
 2. A method for stabilizing a radiotherapeuticcomposition, the method comprising: preparing an ¹⁻³¹I-labeled anti-CD45IgG monoclonal antibody comprising a light chain comprising light chaincomplementarity determining regions 1 through 3 respectively having theamino acid sequences set forth in SEQ ID NO:3, SEQ ID: NO:4 and SEQ IDNO:5 and a heavy chain comprising heavy chain complementaritydetermining regions 1 through 3 respectively having the amino acidsequences set forth in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO: 8, andsupplementing the ¹³¹I-labeled anti-CD45 IgG monoclonal antibody with0.5-5.0% (w/v) ascorbic acid and 0.5-5.0% (w/v) polyvinylpyrrolidone(PVP) to form the radiotherapeutic composition.
 3. A method forpreparation of a stabilized radiotherapeutic composition, the methodcomprising: preparing an ¹³¹I-labeled anti-CD45 IgG monoclonal antibodycomprising a light chain comprising light chain complementaritydetermining regions 1 through 3 respectively having the amino acidsequences set forth in SEQ ID NO:3, SEQ ID: NO:4 and SEQ ID NO:5 and aheavy chain comprising heavy chain complementarity determining regions 1through 3 respectively having the amino acid sequences set forth in SEQID NO:6, SEQ ID NO:7 and SEQ ID NO: 8; and supplementing the¹³¹I-labeled anti-CD45 IgG monoclonal antibody with 0.5-5.0% (w/v)ascorbic acid and 0.5-5.0% (w/v) polyvinylpyrrolidone (PVP) to form thestabilized radiotherapeutic composition, wherein the stabilizedradiotherapeutic composition is configured as a therapeutic dose foradministration to a human subject having a hematological malignancy, thetherapeutic dose comprising 6.66-45.0 mg of anti-CD45 IgG monoclonalantibody and 100-1500 mCi of the ¹³¹I-labeled anti-CD45 IgG monoclonalantibody, wherein the stabilized radiotherapeutic composition is greaterthan 90% stable for at least four (4) days at −20° C.
 4. The method ofclaim 1, wherein the stabilized radiotherapeutic composition furthercomprises a non-radiolabeled anti-CD45 IgG monoclonal antibody.
 5. Themethod of claim 4, wherein each of the non-radiolabeled anti-CD45 IgGmonoclonal antibody and the ¹³¹I-labeled anti-CD45 IgG monoclonalantibody are the same.
 6. The method of claim 1, wherein the compositionfurther comprises 0.5% to 5.0% (w/v) of HSA.
 7. The method of claim 1,wherein the composition comprises 25 mg/mL ascorbic acid, 20 mg/mL PVP,and 50 mM sodium phosphate buffered saline (0.9%) (pH 7.0).
 8. Themethod of claim 2, wherein the radiotherapeutic composition comprises 25mg/mL ascorbic acid, 20 mg/mL PVP, and 50 mM sodium phosphate bufferedsaline (0.9%) (pH 7.0).
 9. The method of claim 3, wherein thehematologic malignancy is selected from the group consisting of acutemyeloid leukemia, myelodysplastic syndrome, acute lymphoblasticleukemia, Hodgkin's disease and non-Hodgkin's lymphoma.
 10. The methodof claim 2, wherein the stabilized radiotherapeutic composition furthercomprises a non-radiolabeled anti-CD45 IgG monoclonal antibody.
 11. Themethod of claim 1, wherein the subject is afflicted with a hematologicalmalignancy.
 12. The method of claim 11, wherein the hematologicmalignancy is selected from the group consisting of acute myeloidleukemia, myelodysplastic syndrome, acute lymphoblastic leukemia,Hodgkin's disease and non-Hodgkin's lymphoma.
 13. The method of claim11, wherein the hematological malignancy is acute myeloid leukemia, andthe subject is a human subject with relapsed or refractory acute myeloidleukemia and at least 55 years old.
 14. The method of claim 5, whereinthe stabilized radiotherapeutic composition comprises a therapeutic doseof 6.66-45.0 mg of anti-CD45 IgG monoclonal antibody and 100-1500 mCi ofthe ¹³¹I-labeled anti-CD45 IgG monoclonal antibody.
 15. The method ofclaim 14, wherein the therapeutic dose does not exceed a ratio of 30:1of mCi ¹³¹1 to mg of the anti-CD45 IgG monoclonal antibody.
 16. Themethod of claim 5, wherein the stabilized radiotherapeutic compositioncomprises a patient specific therapeutic dose of 0.5 mg/kg patientweight anti-CD45 IgG monoclonal antibody and 100-1500 mCi ¹³¹I-labeledof the anti-CD45 IgG monoclonal antibody.
 17. The method of claim 10,wherein each of the non-radiolabeled anti-CD45 IgG monoclonal antibodyand the ¹³¹I-labeled anti-CD45 IgG monoclonal antibody are the same. 18.The method of claim 17, wherein the radiotherapeutic compositioncomprises a therapeutic dose of 6.66-45.0 mg of anti-CD45 IgG monoclonalantibody and 100-1500 mCi ¹³¹I-labeled of the anti-CD45 IgG monoclonalantibody.
 19. The method of claim 2, wherein the radiotherapeuticcomposition is configured as a therapeutic dose for administration to asubject having a hematological malignancy selected from the groupconsisting of acute myeloid leukemia, myelodysplastic syndrome, acutelymphoblastic leukemia, Hodgkin's disease and non-Hodgkin's lymphoma.20. The method of claim 19, wherein the hematological malignancy isacute myeloid leukemia, and the subject is a human subject with relapsedor refractory acute myeloid leukemia and at least 55 years old.
 21. Themethod of claim 1, wherein the heavy chain further comprises an asparticacid residue at position
 141. 22. The method of claim 2, wherein theheavy chain further comprises an aspartic acid residue at position 141.23. The method of claim 3, wherein the heavy chain further comprises anaspartic acid residue at position 141.